Methods of maintaining, expanding, and differentiating neuronal subtype specific progenitors

ABSTRACT

Methods for expanding proliferating populations of neuronal subtype-specific progenitors and creating substantially pure populations of motor neurons are provided herein. In particular, the present invention provides methods for maintaining the unique gene profile and differentiation potential of neuronal subtype-specific progenitors, such as motor neuron progenitors and hindbrain serotonergic neural progenitors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/726,121, filed Oct. 5, 2017, which is a divisional of U.S.application Ser. No. 15/016,934, filed Feb. 5, 2016, which is acontinuation-in-part claiming the benefit of U.S. ProvisionalApplication No. 62/112,441, filed Feb. 5, 2015 and U.S. application Ser.No. 14/194,130, filed Feb. 28, 2014, which claims the benefit of U.S.Provisional Patent Application No. 61/771,572, filed Mar. 1, 2013, eachof which is incorporated herein by reference as if set forth in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under NS074189 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates to methods of expanding the population ofneuronal subtype specific progenitors differentiated from humanpluripotent stem cells, such as spinal motor neuron progenitors andhindbrain serotonergic neuron progenitors. In particular, the presentinvention relates to methods of maintaining the regional identity anddifferentiation potential of neuronal subtype specific progenitorsduring expansion.

BACKGROUND OF THE INVENTION

The mammalian central nervous system is a complex neuronal networkconsisting of a diverse array of cellular subtypes generated in aprecise spatial and temporal pattern throughout development. Eachneuronal subtype within a particular region of the brain and spinal cordcarries a unique set of neurotransmitters and establishes connectionswith its own targets. It is the diversity in molecular and morphologicalcharacteristics of neurons which underlies neural circuit formation.

Extrinsic signals provide neuronal progenitors in the forming neuraltube with positional identity, such that distinct types of neuronalprogenitors express a unique combination of transcription factors. Thistranscriptional code determines neural progenitor identity. Asprogenitors differentiate, they generate distinct neuronal subtypes thatare also characterized by transcriptional codes and secretion ofspecific transmitters. For example, motor neurons (MNs) are a highlyspecialized class of neurons that reside in the spinal cord and projectaxons in organized and discrete patterns to muscles to control theiractivity. Motor neurons secrete the transmitter acetylcholine, expresstranscription factors including MNX1 (also known as HB9), ISL1, andLHX3, and are derived from motor neuron progenitors which express thebasic helix-loop-helix (bHLH) transcription factor OLIG2. Duringneurogenesis, OLIG2 is expressed by MNPcells and is required for thegeneration of MNs, while the homeodomain protein NKX2.2 is expressed inp3 progenitors and induces V3 neurons. Dessaud et al., Development135:2489-2503 (2008). The most prominent MN diseases are spinal muscularatrophy (SMA) and amyotrophic lateral sclerosis (ALS), in which MNsperish in the disease. For review, see Kanning et al., Annu. Rev.Neurosci. 33:409-410 (2010). Similarly, hindbrain serotonin neuronalprogenitors express NKX2.2 together with GATA2 but not OLIG2 or PHOX2band generate serotonin-secreting neurons that project to the entirebrain and spinal cord. Numerous psychiatric disorders involvedysfunctional serotonin neurons. For review, see Gordis & Rohrer, Nat.Rev. Neurosci. Vol. 3(7):531-541 (2002); Kiyasova & Gaspar, Eur. J.Neurosci. Vol. 34(10):1553-1562, (2011).

Neural progenitor cells have been expanded in culture in the presence ofmitogens such as epidermal growth factor (EGF) and/or fibroblast growthfactor 2 (FGF2). For review, see Weiss et al., Trends Neurosci. Vol.19:387-393 (1996). Neural progenitors expanded under such conditionsexhibit diminished potential for generating neurons over glial cells.See Temple, Nature Vol. 414:112-117 (2001). This trend is in generalagreement with the shift from neurogenesis to gliogenesis observedduring normal development. Embryonic ventral mesencephalic progenitors,which produce robust dopaminergic neurons at the time of isolation, losetheir dopaminergic potential shortly after expansion in the presence ofFGF2. See Studer et al., Nat. Neurosci. Vol. 1:290-295 (1998).Similarly, human embryonic stem cell (ESC)-derived neural progenitorsretain their positional identity, as determined by homeodomaintranscription factor expression, and a high degree of neurogenicpotential even after months of expansion. See Zhang et al., J.Hematother. Stem Cell Res. Vol. 12:625-634 (2003). The potential toproduce large projection neurons such as midbrain dopamine neurons,spinal cord motor neurons, and hindbrain serotonergic neurons, however,fades within two to four passages and is replaced by other neuronalpopulations. This phenomenon creates a barrier for producing consistentpopulations of neuronal progenitors with predictable differentiationpotential and functional properties. Accordingly, there remains a needfor compositions and methods for expanding neuronal progenitors whilemaintaining the differentiation potential of the progenitors to yieldthe predicted array of diverse neuronal subtypes.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for maintaining apopulation of neuronal subtype-specific progenitors. Typically, themethod does not require cell sorting. The method can comprise culturingneuronal subtype-specific progenitors in a culture medium comprising aWnt signaling pathway agonist, an inhibitor of the Bone MorphogeneticProtein (BMP) signaling pathway, an inhibitor of the transforming growthfactor beta (TGFβ) signaling pathway, and a Notch signaling pathwayagonist whereby expression of a neuronal subtype-specific progenitorgene expression profile is maintained in the neuronal subtype-specificprogenitors. The neuronal subtype-specific progenitors can have a geneexpression profile comprising expression of at least one of SOX1, SOX2,NESTIN, N-Cadherin, and Ki67. The neuronal subtype-specific progenitorscan be spinal neural progenitors having a gene expression profilefurther comprising expression of at least one of HOXA5 and HOXB8, andsubstantially no expression of midbrain, hindbrain, or forebrainmarkers. The spinal neural progenitors can be OLIG2⁺ spinal motor neuronprogenitors.

The neuronal subtype-specific progenitors can be hindbrain neuralprogenitors having a gene expression profile further comprisingexpression of at least one of GBX2, KROX20, HOXA1-4, and HOXB1-4, andsubstantially no expression of forebrain, spinal cord, or midbrainmarkers. The hindbrain neural progenitors can be NKX2.2⁺ hindbrainserotonergic neural progenitors.

In some cases, the neuronal subtype-specific progenitors are midbrainneural progenitors having a gene expression profile further comprisingexpression of at least one of EN1 and EN2, and substantially noexpression of forebrain, spinal cord, or hindbrain markers. The midbrainneural progenitors can be LMX1A⁺ midbrain dopaminergic neuronprogenitors.

The neuronal subtype-specific progenitors can be forebrain neuralprogenitors having a gene expression profile further comprisingexpression of at least one of FOXG1 and OTX2, and substantially noexpression of midbrain, spinal cord, or hindbrain markers. The forebrainneural progenitors can be NKX-2.1⁺ forebrain GABAergic neuronprogenitors.

The Wnt signaling pathway agonist can be a GSK3 inhibitor selected fromthe group consisting of CHIR99021 and 6-bromo-iridium-3′-oxime. The BMPsignaling pathway inhibitor can be selected from the group consisting ofDMH-1, Dorsomorphin, and LDN-193189. The Notch signaling pathway agonistcan be a histone deacetylase (HDAC) inhibitor selected from the groupconsisting of valproic acid (VPA), suberoyl bis-hydroxamic acid (SAHA),and sodium butyrate. The TGFβ signaling pathway inhibitor can beselected from the group consisting of SB431542, SB505124, and A83-01.The culture medium can comprise CHIR99021, DMH-1, SB431542, and VPA. Theculture medium can comprise between about 1 μM-3 μM CHIR99021; about 1μM-5 μM DMH-1; about 1 μM-5 μM SB431542; and about 0.2-mM-2 mM VPA.

The neuronal subtype specific progenitors can be OLIG2⁺ spinal motorneuron progenitors, where the culture medium comprises CHIR99021, DMH-1,SB431542, VPA, a SHH pathway agonist, and a RA pathway agonist. The SHHpathway agonist can be selected from the group consisting ofpurmorphamine and SAG (Smoothened Agonist). The RA pathway agonist canbe retinoic acid. The culture medium can comprise between about 1 μM to3 CHIR99021; about 1 μM to 5 μM DMH-1; about 1 μM to 5 μM SB431542;about 0.2 mM-2 mM VPA; and about 0.1 μM to 1 μM purmorphamine; about0.01 μM to 1 μM RA. The OLIG2⁺ spinal motor neuron progenitors can bemaintained in a culture substantially free of MNX1⁺ post-mitotic motorneurons for at least 5 weeks. The OLIG2⁺ spinal motor neuron progenitorscan be maintained in a culture substantially free of MNX1⁺ post-mitoticmotor neurons for at least 10 weeks.

The neuronal subtype specific progenitors can be NKX2.2⁺ hindbrainserotonergic neural progenitors, where the culture medium comprisesCHIR99021, DMH-1, SB431542, VPA, and purmorphamine. The culture mediumcan comprise about 1 μM to 3 μM CHIR99021; about 1 μM to 5 μM DMH-1;about 1 μM to 5 μM SB431542; about 0.2 mM-2 mM VPA; and about 0.1 μM to1 μM purmorphamine. The NKX2.2⁺ hindbrain serotonergic neuralprogenitors can be maintained substantially free from differentiationfor at least 5 weeks. The NKX2.2⁺ hindbrain serotonergic neuralprogenitors can be maintained substantially free from differentiationfor at least 10 weeks.

In some cases, neuronal subtype specific progenitors are obtained frompluripotent stem cells. The pluripotent stem cells can be humanpluripotent stem cells. The human pluripotent stem cells can be humanembryonic stem cells or human induced pluripotent stem cells. Theneuronal subtype specific progenitors can be obtained from a humanembryo.

In another version, the present invention is a method of generatingpopulations of motor neuron progenitor cells from stem cells, comprisingthe steps of (a) culturing pluripotent stem cells in a culture mediumcomprising a Wnt signaling pathway agonist, a BMP signaling pathwayinhibitor, and a TGFβ signaling inhibitor, wherein a population of atleast at least 90%, preferably 95%, more preferably 98%, pureSox1+/Hoxa3+ neural stem cells is obtained, and (b) culturing the neuralstem cells of step (a) in a medium additionally comprising retinoic acidand purmorphamine, wherein a population of at least 85%, preferably 90%,more preferably 95%, pure Olig2+/Nkx2.2− motor neuron progenitor cellsis obtained.

In a preferred version, the concentration of the Wnt signaling pathwayagonist is decreased in step (b) relative to step (a). In anotherpreferred version, the Wnt signaling pathway agonist is a GSK3 inhibitorselected from the group consisting of CHIR99021 and6-bromo-iridium-3′-oxime. In a preferred version, the BMP signalingpathway inhibitor is selected from the group consisting of DMH-1,Dorsomorphin, and LDN-193189. In a preferred version, the TGFβ signalinginhibitor is selected from the group consisting of SB431542, SB505124,and A83-01. In a preferred version, the concentration of Wnt signalingpathway agonist CHIR99021 is 3 (+/−10%) in step (a) and 1 (+/−10%) instep (b).

In a preferred version, the method further comprises step (c), culturingthe motor neuron progenitor cells of step (b) in a medium comprisingretinoic acid, purmorphamine and a Notch signaling inhibitor, wherein apopulation of at least 80% pure MNX1+/ChAT+ motor neurons is obtained.Preferably, the Notch inhibitor is selected from the group consisting ofCompound E, DAPT, and DBZ.

In a preferred method, a single MNP has been expanded by a factor of atleast 1×10⁴.

In another version of the invention, the method further comprises thestep of culturing the motor neuron progenitor cells of step (b) in amedium comprising retinoic acid, purmorphamine and a Notch signalinginhibitor, wherein a population of at least 80%, preferably at least85%, more preferably at least 90% pure MNX1+/ChAT+ motor neurons isobtained.

In another version, the present invention is a population of cells asdescribed above. In one version of the invention, the population isSox1+/Hoxa3+ caudal neural stem cells obtained by the method describedabove. In another version, the population is Olig2+/Nkx2.2-motor neuronprogenitor cells obtained by the method described above. In anotherversion, the population is MNX1+/ChAT+ motor neuron cells obtained bythe method described above. Preferably, these cells exhibit formation ofneuromuscular junctions when co-cultured with skeletal muscle cells andprojection of axons toward muscles when grafted into the developingchick spinal cord.

These and other features, objects, and advantages of the presentinvention will become better understood from the description thatfollows. In the description, reference is made to the accompanyingdrawings, which form a part hereof and in which there is shown by way ofillustration, not limitation, embodiments of the invention. Thedescription of preferred embodiments is not intended to limit theinvention to cover all modifications, equivalents and alternatives.Reference should therefore be made to the claims recited herein forinterpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing in color.Copies of this patent or patent application publication with colordrawings will be provided by the Office upon request and payment of thenecessary fee.

FIG. 1 is a flow chart depicting differentiation of spinal motor neuronprogenitors and hindbrain serotonergic neuron progenitors frompluripotent stem cells and expansion of these neuronal progenitors underspecified conditions. Abbreviations: PSC (pluripotent stem cell); NE andNEP (neuroepithelial progenitor); MNP (motor neuron progenitor); SNP(serotonergic neural progenitor); RA (retinoic acid); MN (motor neuron).The curved arrows indicate an expanded or maintained population.

FIG. 2 is a flow chart depicting an exemplary protocol fordifferentiating mature motor neurons from an expanded population ofspinal motor neuron progenitors. MN (motor neuron.)

FIG. 3 is a flow chart of an improved method of generating motorneurons. Cmpd E (Compound E, as described below.)

FIGS. 4A-4D disclose generation of pure population of OLIG2⁺ MNPs fromhPSCs. (FIG. 4A) Schematics showing the time course and small moleculecocktail for hPSC differentiation into MNPs. (FIG. 4B) The regionalidentity (OTX2+vs. HOXA3+) and quantitation of SOX1+ NEPs after 6 daysof culture in CHIR+SB+DMH-1 vs. SB+DMH-1 condition. Scale bars: 50 μm.(FIG. 4C) CHIR+SB+DMH-1+RA+Pur cocktail induced pure MNPs at Day 12,which express OLIG2 (green) but not NKX2.2 (red). Quantification isshown on right. Scale bars: 50 μm. (FIG. 4D) The efficiency of OLIG2⁺MNP differentiation from multiple hPSC lines.

FIGS. 5A-5E show the expansion of OLIG2⁺ MNPs. (FIG. 5A) RA and Pur arerequired for maintaining the identity of MNPs with OLIG2 (green)expression, by preventing a switch to NKX2.2⁺ (red) progenitors. Scalebars: 50 μm. (FIG. 5B) CHIR+SB+DMH-1 are required for maintaining theproliferation of MNPs with Ki67 (red) expression at the maximum level.Scale bars: 50 μm. (FIG. 5C) Schematics showing the expansion of MNPswith the combination of small molecules. (FIG. 5D) The MNPs wereexpanded for at least 5 passages yet maintained the OLIG2 (green)expression. (FIG. 5E) Cumulative hPSC-derived MNP counts over fivepassages (passages denoted p1-p5).

FIGS. 6A-6D present MNPs differentiating into enriched functional MNs.(FIG. 6A) Schematics showing the time course and small molecule cocktailfor MNP differentiation into mature MNs. (FIG. 6B) Quantification ofMNX1⁺ and ISL1⁺ (green) MNs on Day 6, and CHAT⁺ (red) mature MNs on Day16. Scale bars: 50 μm. (FIG. 6C) MNs, stained with CHAT antibody (red),formed neuromuscular junctions, labeled with bungarotoxin (BTX, green),when co-cultured with myotubes. Scale bars: 100 μm. (FIG. 6D)Representative image of xenotransplantation of GFP labeled human MNsinto a developing chicken embryo. Scale bars: 50 μm. (FIG. 6D′)magnification of the field showing that human MN axons (GFP⁺/CHAT⁺)projected ventrally through the ventral roots.

FIGS. 7A-7D show enriched MNs enable presentation of disease phenotypesand building of screening platforms. (FIG. 7A) SMA MNs exhibited a lower(38±4%) proportion of full-length SMN among total SMN mRNA than that inSMA GABA neurons (60±6%), when comparing to wild type (WT) neurons(*p<0.05). (FIG. 7B) ALS (D90A) MNs exhibited 45±4% reduction of NEFLmRNA than that in ALS GABA neurons, when comparing to corrected (D90D)neurons (*p<0.05). (FIG. 7C) ALS (D90A) MNs showed neurite fragmentationand reduced neurite length when culturing on ALS (D90A) astrocytes,comparing to that on corrected (D90D) astrocytes. Scale bars: 50 μm.(FIG. 7D) Schematic of SYP-Nluc reporter. (FIG. 7E) Quantification ofNluc activity (left panel) and ratio (right panel) of SYP-Nluc reporterMNs on ALS (D90A) and corrected (D90D) astrocytes, when comparingbetween the control, Riluzole (Rilu), Kenpaullone (Ken) and EphAinhibitor (EphAi) groups (** P<0.01).

FIGS. 8A-8B are the serial titration of Pur and CHIR concentration. Incombination with 0.1 μM RA, 2 μM SB and 2 μM DMH-1, Pur concentrationvaried from 0.12 to 1 μM (FIG. 8A), and CHIR concentration varied from 1μM to 3 μM (FIG. 8B). OLIG2⁺ MNPs were quantified under differentconditions.

FIGS. 9A-9E disclose the electrophysiological characteristics of MNs.(FIG. 9A) Whole-cell patch-clamp recording on a neuron cultured onastrocytes for 28 days. (FIG. 9B) Electrophysiological characteristics,including capacitance, resting membrane potential (RMP) and resistance(Rin). (FIG. 9C) Inward Na+ and outward K+ currents were triggered upon−50 mV to 50 mV voltage steps. (FIG. 9D) Action potentials were inducedfrom −40 pA to 100 pA injected current steps. (FIG. 9E)Immunocytochemistry reveals the recorded cells (identified by biocytinback-fill) to be CHAT positive.

FIGS. 10A-10C show the generation of MNs and GABA+ non-MNs from humaniPSCs. (FIG. 10A) Schematics showing the time course and small moleculecocktail for hiPSCs differentiation into MNs and GABA neurons. Cyc is anSHH antagonist. Repression of SHH signaling induces the dorsal GABAneuron. Immunofluorescent images of GABA⁺ neurons (FIG. 10B) and CHAT⁺MNs (FIG. 10C) on Day 28. Scale bars: 50 μm. The term CSD stands forthree small molecules—CHIR99021, SB431542, and DMH1.

FIG. 11 shows the ALS mutant and corrected astrocytes. Immunofluorescentimages of ALS mutant (D90A) and corrected (D90D) astrocytesdifferentiated from hiPSCs for 6 months. Scale bars: 50 μm.

FIGS. 12A-12B disclose the SYP-Nluc reporter assay. (FIG. 12A) Thelinear relationship between Nluc activity and MN number. (FIG. 12B) TheNluc activity of SYP-Nluc MNs was lower when co-cultured on D90Aastrocytes, comparing to that on D90D astrocytes (***P<0.001).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the inventors'discovery that a defined cocktail of small molecules or chemicalcompounds could be used to maintain the proliferation of neuronalsubtype-specific progenitor cells, such as spinal motor neuronprogenitors and hindbrain serotonergic neuron progenitors differentiatedfrom human pluripotent stem cells. The inventors further discovered thatcertain culture conditions could maintain in vitro cultured neuronalsubtype-specific progenitor cells in their progenitor state withsubstantially no loss of differentiation potential. Upon providing adifferentiation condition to the maintained, expanded progenitors, theInventors induced differentiation of the progenitors into, for example,mature motor neurons and serotonergic neurons.

Methods of Generating and Maintaining Neuronal Subtype-SpecificProgenitors

In one aspect, therefore, the present invention is directed to methodsfor generating and maintaining a population of neuronal subtype specificprogenitors. Neuronal subtype-specific progenitors can include, withoutlimitation, forebrain neural progenitors, spinal neural progenitors,hindbrain neural progenitors, and midbrain neuron progenitors. Thephenotype of a neuronal subtype specific progenitor is specified by theexpression of unique combination of transcription factors inrostral-caudal and dorsal-ventral patterns. For example, forebrainneural progenitors can be NKX-2.1⁺ forebrain GABAergic neuronprogenitors, and midbrain neural progenitors can be LMX1A⁺ midbraindopaminergic neuron progenitors.

A method for generating a population of neuronal subtype-specificprogenitors can include culturing neuroepithelial cells in a culturemedium comprising a Wnt signaling pathway agonist, an inhibitor of thebone morphogenetic protein (BMP) signaling pathway, an inhibitor of thetransforming growth factor beta (TGFβ) signaling pathway, and Notchsignaling pathway agonist, and at least one of retinoic acid (RA) or asonic hedgehog (SHH) pathway agonist, where the cells are cultured for atime sufficient to induce expression of a neuronal subtype-specificprogenitor gene expression profile. A neuronal subtype-specific geneexpression profile will include expression of at least one of SOX1,SOX2, NESTIN, N-Cadherin, and Ki67. In the case of hindbrain neuralprogenitors, the gene expression profile can further include at leastone of GBX2, KROX20, HOXA1-4, and HOXB1-4, but substantially noexpression of forebrain, spinal cord, or midbrain markers. In anexemplary embodiment, a hindbrain neural progenitor is a NKX2.2⁺hindbrain serotonergic neural progenitor. For midbrain neuralprogenitors, the gene expression profile can further include at leastone of EN1, LMX1A, and LMX1B but substantially no expression offorebrain, spinal cord, or hindbrain markers. In some cases, a midbrainneural progenitor is a LMX1A⁺ midbrain dopaminergic neuron progenitor.For forebrain neural progenitors, the gene expression profile canfurther include at least one of FOXG1, OTX2, EMX1, NKX2.1, and SIX3, butsubstantially no expression of midbrain, spinal cord, or hindbrainmarkers. In some cases, the forebrain neural progenitor is a NKX-2.1⁺forebrain GABAergic neuron progenitor. For a spinal neural progenitor,the gene expression profile can further include at least one of HOXB6and HOXB8, but substantially no expression of midbrain, hindbrain, orforebrain markers. In some cases, the spinal neural progenitor is aOLIG2⁺ spinal motor neuron progenitor.

In some cases, a method for generating neuronal subtype-specificprogenitors can further comprise culturing pluripotent stem cells in aculture medium for a time sufficient to induce differentiation of thepluripotent stem cells into neuroepithelial cells. The culture mediumcan comprise (i) a Wnt signaling pathway agonist, (ii) an inhibitor ofthe BMP signaling pathway, and (iii) an inhibitor of the TGFβ signalingpathway. Pluripotent stem cells that can be used include humanpluripotent stem cells such as human embryonic stem cells and humaninduced pluripotent stem cells.

Methods of maintaining a population of neuronal subtype specificprogenitors derived from pluripotent stem cells can comprise culturingneuronal subtype specific progenitors in a culture medium comprising aWnt signaling pathway agonist, an inhibitor of the bone morphogeneticprotein (BMP) signaling pathway, an inhibitor of the transforming growthfactor beta (TGFβ) signaling pathway, and a Notch signaling pathwayagonist. In some cases, such a culture medium is called a maintenanceculture medium. By “maintaining” a population of neuronalsubtype-specific progenitors, we mean maintenance of a phenotype of aunique gene expression profile (e.g., profile of transcription factorsexpressed in a given cell type) characteristic of a given neuronalsubtype specific progenitor. As used herein, the term “maintaining”refers to maintenance of such a phenotype (e.g., cell morphology, geneexpression profile) characteristic of a given neuronal subtype specificprogenitor for at least 5 passages or at least 5 weeks, preferably atleast 8 passages or at least 8 weeks, and most preferably at least 10passages or at least 10 weeks.

A culture medium comprising small molecule agonists of each of the Wntand Notch signaling pathways, and small molecule inhibitors of thetransforming growth factor beta (TGFβ) and BMP pathways is required formaintaining the proliferation and self-renewal of neuronal progenitorsgenerally. However, other small molecules or patterning factors areadditionally required for maintaining the unique gene expression profilecharacteristic of a neuronal subtype specific progenitor. For example, aSonic Hedgehog (SHH) signaling pathway agonist (e.g., purmorphamine) anda retinoic acid (RA) signaling pathway agonist are additionally requiredto maintain expression of the transcription factor OLIG2 in motor neuronprogenitors and to maintain motor neuron progenitor identity anddifferentiation capacity. Similarly, a SHH signaling pathway agonist isadditionally required to maintain expression of the transcription factorNKX2.2 in hindbrain serotonergic neuron progenitors and to maintainhindbrain serotonergic neuron progenitor identity and differentiationcapacity.

In an exemplary embodiment, a culture medium for maintaining apopulation of any type of other neuronal subtype specific progenitorsaccording to a method provided herein comprises RA, purmorphamine, theGSK3 inhibitor CHIR99021, the BMP signaling inhibitor DMH-1, and theTGFβ signaling inhibitor SB431542. In some cases, the culture mediumfurther comprises between about 0.1 μM to 1.0 μM RA, and between about0.1 μM to 1.0 μM purmorphamine.

In other cases, maintaining neuronal progenitors according to a methodprovided herein can include providing the cells with a culture mediumcomprising an agonist of Notch signaling such as, for example, VPA(Valproic acid). VPA is available from several commercial chemicalcompound vendors (e.g., Tocris Bioscience, Sigma-Aldrich). VPA is anHDAC inhibitor which can indirectly activate Notch signaling.Stockhausen et al., Br. J. Cancer. Vol. 92(4):751-759 (2005). Othersmall molecule inhibitors of HDAC which can be used to activate Notchsignaling include, for example, suberoyl bis-hydroxamic acid (SAHA) andsodium butyrate. Accordingly, a culture medium appropriate for use in amethod for maintaining neuroepithelial cells can comprise CHIR99021,DMH-1, SB431542, and at least one of valproic acid (VPA), a SHH pathwayagonist, and RA (or RA pathway agonist). In some cases, the culturemedium can comprise between about 1 μM-3 μM CHIR99021, between about 1μM-5 μM DMH-1, between about 1 μM-5 μM SB431542, and at least one ofbetween about 0.2 mM-2 mM VPA, between about 0.2 μM-2 μM RA, and betweenabout 0.2 μM-2 μM purmorphamine.

Any appropriate culture method can be used to practice a method providedherein. In an exemplary embodiment, adherent culture methods can beused. Adherent culture (or “colony culture”) allows direct visualizationof neural differentiation, including the formation of neural tube-likerosettes during neuroepithelial induction and the migration ofneuroepithelial cells. Adherent/colony culture permits ready removal ofnon-neural colonies and promotes subsequent neural differentiation. Insome cases, suspension culture can be used for initially separatingpluripotent cells from mouse embryonic fibroblast (MEF) feeder cells orfor purifying neuroepithelial cells.

Methods of Generating and Maintaining Motor Neuron Progenitors

In another aspect, the present invention is directed to methods forgenerating motor neuron progenitors and methods for maintaining anexpanded population of motor neuron progenitors. As used herein, theterm “motor neuron progenitor” refers to a progenitor or precursor cellwhich will mature, or is capable of maturing, into a motor neuron,wherein the MNPs express Olig2 but do not express NkX2.2.

To generate motor neuron progenitors, a first step in the method can beto generate a population of neuroepithelial cells. Neuroepithelial cellsare also known as neural stem cells, and the terms “neuroepithelialcell” and “neural stem cell” are used interchangeably throughout. Amethod for generating a population of motor neuron progenitors cancomprise culturing human pluripotent stem cells in a culture mediumcomprising a Wnt signaling pathway agonist, an inhibitor of the BMPsignaling pathway, and an inhibitor of the TGFβ signaling pathway for atime sufficient to induce differentiation of pluripotent stem cells intoneuroepithelial cells. Pluripotent stem cells useful for the methodsprovided herein include human embryonic stem cells (hESCs) and humaninduced pluripotent stem cells (hIPS cells).

In some cases, a culture medium appropriate for generating a populationof neuroepithelial cells can comprise a plurality of small molecules orother chemical compounds which promote the differentiation ofpluripotent stem cells into neuroepithelial cells. In some cases, such aculture medium is called a differentiation culture medium. The pluralityof small molecules or chemical compounds can include an agonist of thecanonical Wnt signaling pathway, an inhibitor of the BMP signalingpathway, and an inhibitor of TGFβ signaling. For example, a method forgenerating a population of neuroepithelial cells can include providingpluripotent stem cells with a culture medium comprising CHIR99021, aGSK3 inhibitor. By inhibiting GSK3, CHIR99021 activates the canonicalWnt signaling pathway. CHIR99021 has been reported to inhibit thedifferentiation of mouse and human embryonic stem cells (ESCs) throughWnt signaling. For review, see Wray and Hartmann, Trends in Cell Biology22:159-168 (2012). Another GSK3 inhibitor which can be used is, forexample, the Wnt/β-catenin signaling agonist 6-bromo-iridium-3′-oxime(“BIO”). See Meijer et al., Chem. Biol. 10(12):1255-66 (2003). GSK3inhibitors such as those described herein are available from commercialvendors of chemical compounds (e.g., Selleckchem, Tocris Bioscience).

In some cases, an inhibitor of BMP signaling is DMH-1, which blocks BMPsignaling by inhibiting Activin receptor-like kinase (ALK2). Other smallmolecule inhibitors of Activin receptor-like kinases which can be usedto block BMP signaling include, for example, Dorsomorphin andLDN-193189. Both compounds affect Smad-dependent and Smad-independentBMP signaling triggered by BMP2, BMP6, or GDFS. Boergermann et al., Int.J. Biochem. Cell Biol. 42(11): 1802-7 (2010).

In some cases, an inhibitor of TGFβ signaling is SB431542, whichinhibits Activin receptor-like kinases 4, 5, and 7 (ALK4, ALK5, andALK7). SB431542 can be purchased from any one of several commercialchemical compound vendors (e.g., Tocris Bioscience, Sigma-Aldrich). Byinhibiting Activin receptor-like kinases 4, 5, and 7, SB431542 inhibitsTGFβ signaling. Other small molecule inhibitors of Activin receptor-likekinase 5 (ALK5) (also known as transforming growth factor-α type Ireceptor kinase) such as SB505124 and A83-01 can be used to inhibitTGFβsignaling.

In an exemplary embodiment, a culture medium for use according to amethod provided herein comprises the GSK3 inhibitor CHIR99021, the BMPsignaling inhibitor DMH-1, and the TGFβ signaling inhibitor SB431542. Insome cases, the culture medium can comprise between about 1 μM-3 μMCHIR99021, between about 1 μM-5 μM DMH-1, and between about 1 μM-5 μMSB431542.

In some cases, a culture medium for use according to a method providedherein comprises a basal culture medium supplemented with smallmolecules or chemical compounds such as those described herein. Forexample, a culture medium can be Neurobasal® culture medium (LifeTechnologies. In some cases, a culture medium comprises DMEM/F12,Neurobasal medium at 1:1, 1×N2 neural supplement (N-2 Supplement;Gibco), 1× B27 neural supplement (B-27 Supplement; Gibco), and 1 mMascorbic acid.

A method for generating motor neuron progenitors can further compriseinducing neuroepithelial cells to differentiate into spinal motor neuronprogenitors. In some cases, the method comprises culturingneuroepithelial cells in a culture medium comprising a Wnt signalingpathway agonist, an inhibitor of the BMP signaling pathway, an inhibitorof the TGFβ signaling pathway, a sonic hedgehog (SHH) signaling agonist,and a RA signaling agonist for a time sufficient to induce expression ofa motor neuron progenitor marker (OLIG2).

In some cases, generating motor neuron progenitors according to a methodprovided herein can include providing neuroepithelial cells (e.g., stemcell-derived NE cells) with a culture medium comprising a SHH signalingpathway agonist such as, for example, purmorphamine. Purmorphamine isavailable from several commercial chemical compound vendors (e.g.,Tocris Bioscience, Stemgent). Purmorphamine activates SHH signaling bydirectly targeting Smoothened (“Smo”), a critical component of the SHHsignaling pathway. Sinha et al., Nature Chem. Biol. 2:29-30 (2006).Other small molecule agonists of Smo which can be used to activate SHHsignaling include, for example, SAG (“Smoothened Agonist”). The hedgehogpathway agonist SAG is a cell-permeable chlorobenzothiophene compoundthat modulates the coupling of Smo with its downstream effector byinteracting with the Smo heptahelical domain. SHH acts in a gradedmanner to establish different neural progenitor cell populations. SeeBriscoe et al., Semin. Cell Dev. Biol. 10(3):353-62 (1999).

In some cases, generating motor neuron progenitors according to a methodprovided herein can include providing cells with a culture mediumcomprising a RA signaling agonist such as RA (e.g., all trans retinoicacid). RA is available from several commercial chemical compound vendors(e.g., Tocris Bioscience, Sigma-Aldrich). RA activates RA signaling bybinding nuclear hormone receptors retinoic acid receptors (RARs), whichis required for specification of motor neuron progenitors. See Novitchet al., Neuron 40(1):81-95 (2003).

In an exemplary embodiment, a culture medium for generating motor neuronprogenitors according to a method provided herein comprises Wntsignaling agonist CHIR99021, BMP signaling inhibitor DMH-1, TGFβsignaling inhibitor SB431542, SHH signaling agonist purmorphamine, andretinoic acid. In some cases, the culture medium comprises between about1 μM-3 μM CHIR99021, between about 1 μM-5 μM DMH-1, between about 1 μM-5μM SB431542, between about 0.2 μM-2 μM purmorphamine, and between about0.1 μM-1.0 μM RA.

Cells cultured and differentiated according to a method provided hereincan be identified as motor neuron progenitors on the basis of OLIG2⁺expression. The bHLH transcription factor OLIG2 serves as a uniquemarker of MN progenitors. The transcriptional repressor function ofOLIG2 is both necessary and sufficient to stimulate the expression of anumber of downstream homeodomain transcription factors that provide MNswith their unique character. See Briscoe and Novitch, Philos. Trans. R.Soc. Lond. B. Biol. Sci. 363(1489):57-70 (2008); see also Shirasaki andPfaff, Annu. Rev. Neurosci. 25:251-281 (2002).

In some cases, a method provided herein further includes a step ofculturing OLIG2⁺ motor neuron progenitors in a MN progenitordifferentiation culture medium for approximately one week to generateMNX1⁺ post-mitotic motor neurons. MNX1 (also known as Motor Neuron andPancreas Homeobox 1 or HB9) is homeobox gene expressed selectively bymotor neurons in the developing vertebrate central nervous system (Arberet al., Neuron 23(4):659-74 (1999)). Alternatively, post-mitotic motorneurons can be marked by the expression of ISLET1/2. In some cases, amethod provided herein further includes culturing OLIG2⁺ motor neuronprogenitors in a MN progenitor differentiation culture medium for atleast about two weeks (e.g., 2 weeks, 2.5 weeks, 3 weeks) to generatecholine acetyltransferase-positive (ChAr) mature motor neurons. Cholineacetyltransferase is an enzyme that catalyzes the synthesis of thetransmitter acetylcholine for transmitting signals through theneuromuscular junctions and is expressed in somatic, cholinergic(acetylcholine-producing) motor neurons. Mature motor neurons alsoexpress VAChAT (vesicular acetylcholine transporter), a neurotransmittertransporter which is essential for storage of acetylcholine (ACh) insecretory organelles and for release of ACh.

In another aspect, the present invention is directed to methods formaintaining a population of motor neuron progenitors. As used herein,the term “maintaining” refers to maintenance of a phenotype (e.g., cellmorphology, gene expression profile, differentiation potential)characteristic of a given progenitor for at least 5 weeks, preferably atleast 8 weeks, and most preferably at least 10 weeks. For example, thepresent invention provides methods for maintaining OLIG2⁺ motor neuronprogenitors in vitro for at least 5 weeks.

Methods of maintaining a population of motor neuron progenitors derivedfrom pluripotent stem cells can comprise culturing motor neuronprogenitors in a culture medium comprising a Wnt signaling pathwayagonist, an inhibitor of the BMP signaling pathway, an inhibitor of theTGFβ signaling pathway, a Notch signaling pathway agonist, a SHEsignaling pathway agonist, and a RA signaling pathway agonist. In somecases, such a culture medium is called a maintenance culture medium.Maintaining cells according to a method provided herein can includeproviding cells with a culture medium comprising an agonist of Notchsignaling such as, for example, VPA (Valproic acid). VPA is availablefrom several commercial chemical compound vendors (e.g., TocrisBioscience, Sigma-Aldrich). VPA is a histone deacetylase (HDAC)inhibitor which can indirectly activate Notch signaling. Stockhausen etal., Br. J. Cancer. 92(4):751-759 (2005). Other small moleculeinhibitors of HDAC which can be used to activate Notch signalinginclude, for example, suberoyl bis-hydroxamic acid (SAHA) and sodiumbutyrate. Accordingly, a culture medium appropriate for use in a methodfor maintaining motor neuron progenitors can comprise CHIR99021, DMH-1,SB431542, VPA, purmorphamine, and RA.

In an exemplary embodiment, methods for maintaining a population ofmotor neuron progenitors can comprise culturing motor neuron progenitorsin a culture medium comprising between about 1 μM-3 μM CHIR99021;between about 1 μM-5 μM DMH-1; between about 1 μM-5 μM SB431542; betweenabout 0.2 mM-2 mM VPA; between about 0.2 μM-2 μM purmorphamine; andbetween about 0.1 μM-1.0 μM RA. Under these conditions, motor neuronprogenitors maintain long-term OLIG2⁺ expression without differentiatingor switching into other neural progenitor subtypes such as NKX2.2⁺ V3interneuron progenitors (p3 domain progenitors). The motor neuronprogenitors can be maintained for at least 5 weeks (e.g., at least about5 passages), yielding previously unobtainable numbers of MN progenitors(producing on the order of 10⁴ MN progenitors from a single MNprogenitor cell).

Methods of Generating and Maintaining Hindbrain Serotonergic NeuronProgenitors

In a further aspect, the present invention is directed to methods forgenerating and maintaining a population of hindbrain serotonergic neuronprogenitors. The terms “serotonergic neuron progenitor” and“serotonergic neural progenitor” are used interchangeably throughout andrefer to a progenitor or precursor cell which will mature into a neuroncapable of serotonin neurotransmission.

Methods of generating a population of hindbrain serotonergic neuronprogenitors can comprise culturing neuroepithelial cells in a culturemedium comprising a Wnt signaling pathway agonist, an inhibitor of theBMP signaling pathway, and an inhibitor of the TGFβ signaling pathwayplus a SHH signaling pathway agonist for a time sufficient (e.g., about1 week to about 2 weeks) to induce expression of a hindbrain marker.Hindbrain serotonergic neuron progenitors generated from humanpluripotent stem cells according to a method provided herein can bedefined based on their expression of hindbrain markers (e.g., GBX2,KROX20, HOXA1-4, HOXB1-4), but not forebrain markers (e.g., FOXG1, OTX2,EMX1, NKX2.1, SIX3), midbrain markers (e.g., EN1, LMX1A, LMX1B, SIM1,LIM1), or spinal cord markers (e.g., HOXB6, HOXB8) besides the neuralprogenitor markers (e.g., SOX1, SOX2, NESTIN, N-Cadherin, and Ki67).

In an exemplary embodiment, a culture medium for generating a populationof hindbrain serotonergic neuron progenitors according to a methodprovided herein comprises Wnt signaling agonist CHIR99021, BMP signalinginhibitor DMH-1, TGFβ signaling inhibitor SB431542, and SHH signalingpathway agonist purmorphamine. In some cases, the culture medium cancomprise between about 1 μM-3 μM CHIR99021; between about 1 μM-5 μMDMH-1; between about 1 μM-5 μM SB431542; and between about 0.2 μM-2 μMpurmorphamine.

Preferably, the method of the present invention does not require cellsorting.

Methods for maintaining a population of hindbrain serotonergic neuronprogenitors can comprise culturing hindbrain serotonergic neuronprogenitors in a maintenance medium comprising between about 1 μM-3 μMCHIR99021; between about 1 μM-5 μM DMH-1; between about 1 μM-5 μMSB431542; between about 0.2 mM-2 mM VPA, and between about 0.2 μM-2 μMpurmorphamine. Under these conditions, serotonergic neuron progenitorsmaintain long-term NKX2.2⁺ expression without switching into otherneural progenitor subtypes. The serotonergic neural progenitors can bemaintained for at least 5 weeks or at least 5 passages.

Methods of Maintaining Other Neuronal Subtype Specific Progenitors

In a further aspect, the present invention is directed to methods formaintaining a population of any other type of neuronal subtype specificprogenitors, for example, forebrain GABAergic neuron progenitors, ormidbrain dopaminergic neuron progenitors. The phenotype of neuronalsubtype specific progenitors is defined by a unique gene expressionprofile of regional markers and subtype specific markers, and thepotential to differentiate into subtype specific mature neurons, Forexample, forebrain GABAergic neuron progenitor is defined by expressionof forebrain markers FOXG1, OTX2B and subtype specific marker NKX2.1, aswell as its ability to differentiate into mature neuron secreting GABAneurotransmitter. Similarly, a midbrain dopaminergic neuron progenitoris marked by midbrain transcription factors EN1, EN2 and subtypespecific transcription factor LMX1A, as well as its potential todifferentiate into mature neuron secreting dopamine neurotransmitter. By“maintaining” a population of progenitors, we mean maintenance of aphenotype for at least 5 passages or at least 5 weeks, preferably atleast 8 passages or at least 8 weeks, and most preferably at least 10passages or at least 10 weeks.

Methods of maintaining a population of neuronal subtype specificprogenitors can comprise culturing neuronal subtype specific progenitorsin a culture medium comprising a Wnt signaling pathway agonist, aninhibitor of the BMP signaling pathway, an inhibitor of the TGFβsignaling pathway, and a Notch signaling pathway agonist.

The four small molecules, CHIR99021, DMH-1, SB431542, and VPA or theirfunctional equivalents (the core maintaining medium) are required formaintaining the proliferation and phenotype of progenitors. However,other small molecules or patterning factors may be required formaintaining the unique subtype specific progenitors. For example, a SHHsignaling pathway agonist and a RA signaling pathway agonist arerequired in maintaining OLIG2 transcription factor expression in spinalmotor neuron progenitors; a SHH signaling pathway agonist is required inmaintaining NKX2.2 transcription factor expression in hindbrainserotonergic neuron progenitors.

Improved Method of Generating Pure Motor Neuron Progenitors from StemCells

The description of this embodiment of the present invention is based inlarge part on Example 5 but contains method elements disclosed in theprevious embodiments of the present invention, as described above.

As described above, human pluripotent stem cells (hPSCs) have opened newopportunities for understanding early human development, modelingdisease processes and developing new therapeutics. However, some ofthese applications are hindered by low efficiency and heterogeneity oftarget cell types differentiated from hPSCs, such as motor neurons(MNs), as well as the inability to maintain the potency of lineagecommitted progenitors. By using a combination of small molecules thatregulate multiple signaling pathways, we have developed a novel methodto guide hPSCs to a near-pure population (at least 95%) of OLIG2⁺ motorneuron progenitors (MNPs) in 12 days and a highly enriched population(at least 90%) of functionally mature MNs in another 16 days. FIG. 3 isa flow chart of our method with preferred reagent concentrations.

More importantly, we have found that the OLIG2⁺ MNPs can be expanded forat least 5 passages so that a single MNP can be amplified to at least1×10⁴. The MNs produced from the expandable MNPs exhibit functionalproperties, including formation of neuromuscular junctions whenco-cultured with skeletal muscle cells and projection of axons towardmuscles when grafted into the developing chick spinal cord. Theconsistent and highly enriched MN populations enable modeling MNdegenerative diseases and developing large-scale, MN based screeningassays.

The Examples contain specific details of a preferred embodiment. Wedescribe the method in general below:

In general, the present invention is a method of generating populationsof motor neuron progenitor (MNP) cells from stem cells, typically firstcomprising the steps of culturing pluripotent stem cells, as definedabove, into neural stem cells (NSCs) in a culture medium comprising aBMP signaling pathway inhibitor, a Wnt signaling pathway agonist, and aninhibitor of activin-nodal signaling.

We describe above typical examples of BMP signaling pathway inhibitorsand Wnt signaling pathway agonists. Preferably the BMP signaling pathwayinhibitor is selected from the group consisting of DMH-1, Dorsomorphin,and LDN-193189. Most preferably, the inhibitor is DMH1. Typically theWnt signaling pathway agonist is a GSK3 inhibitor selected from thegroup consisting of CHIR99021 and 6-bromo-iridium-3′-oxime. Mostpreferably the agonist is CHIR99021 (CHIR). Preferably, the agonist isused at a concentration of about 3 μM.

The inhibitor of TGFβ signaling is preferably SB431542. (Stemgent).Other suitable inhibitors are described above.

Typically, a population of at least 90%, preferably at least 95%, morepreferably at least 98% pure Sox1+/Hoxa3+ neural stem cells is obtained.

One may then culture the neural stem cells in a medium additionallycomprising retinoic acid and purmorphamine, as described above.Preferably, the concentration of CHIR is between 1-3 preferablydecreased from 3 μM (+/−10%) to 1 μM (+/−10%) relative to the proceedingstep, and a population of at least 85%, preferably at least 90%, morepreferably at least 95%, pure Olig2+/Nkx2.2− motor neuron progenitorcells is obtained.

In another embodiment, the present invention further comprises the stepof culturing the motor neuron progenitor cells in a medium comprisingretinoic acid (0.01-1 μM), purmorphamine (0.01-0.5 μM) and a Notchinhibitor, such as compound E (0.05-0.5 μM, Calbiochem), wherein apopulation, preferably of at least 80%, preferably 85%, more preferably90% pure, MNX1+/ChAT+ motor neurons is obtained.

Other Notch inhibitors (also known as γ-secretase inhibitors) includeDAPT and DBZ.

A preferred timing of cell cultures in the method of the presentinvention is between 5.5 and 6.5 days for pluripotent stem cells todevelop into NSCs and between 5.5 and 6.5 days for NSCs to develop intoMNPs. MNPs are preferably cultured for at least 14-17 more days todevelop into MN cells.

Of course, one may wish to expand or maintain the MNPs, as describedabove, before transformation into MNs using the method described above.

In another embodiment, the present invention is a population ofSox1+/Hoxa3+ neural stem cells obtained by the method described above.

In anther embodiment, the present invention is a population ofOlig2+/Nkx2.2-motor neuron progenitor cells obtained by the methoddescribed above.

In another embodiment, the present invention is a population ofMNX1+/ChAT+ motor neuron cells obtained by the method described above.It is a particular advantage of the present invention that thispopulation can be expanded from a single MNP and, thus, particularlyuseful reagents may be obtained. It is also an advantage that thispopulation exhibits the functional characteristics described above.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar to or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and materials are described herein.

Various exemplary embodiments of compositions and methods according tothis invention are now described in the following non-limiting Examples.The Examples are offered for illustrative purposes only and are notintended to limit the scope of the present invention in any way. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and the following examples and fall within thescope of the appended claims.

EXAMPLES Example 1—Efficient Generation of MN Progenitors from hESCs in2 Weeks

To induce the specification of neuroepithelial cells from humanpluripotent cells, the dual TGFβ/BMP inhibition approach was applied forhuman embryonic stem cells in a monolayer culture. See, for review,Chambers et al., Nature Biotech. 27:275-280 (2009). The small moleculeSB431542 represses TGFβ signaling by selectively inhibiting Activinreceptor-like kinase ALK4/5/7. The small molecule DMH-1 represses BMPsignaling by selectively inhibiting the BMP receptor kinase ALK2. Humanembryonic stem cells (hESCs) were treated with 2 μM DMH-1 and 2 μMSB431542 for 1 week. Treated hESCs were then induced to differentiateinto populations comprising about 85% SOX1⁺ neuroepithelial cells butalso comprising other cell lineages due to spontaneous ESCdifferentiation, since the dual Nodal/BMP inhibitors SB431542 and DMH-1are unable to prevent all spontaneous differentiation into other celllineages, especially when ESC colonies are small. To further improveneural specification, a small molecule that inhibits glycogen synthasekinase-3 (CHIR99021) was applied in combination with DMH-1 and SB431542.GSK3 negatively regulates WNT signaling, and WNT signaling promotes theself-renewal of ESCs and neural progenitors. When exposed to these threemolecules for about 6 days, hESCs not only generated more purepopulations of SOX1⁺ neuroepithelial cells (e.g., at least 95% of cellsin the total population were SOX1⁺ neuroepithelial cells), but alsogenerated 2.5-fold more neuroepithelial cells. However, CDS (CHIR99021,DMH-1, and SB431542) treatment-derived neuroepithelial cells showedcaudal identity as demonstrated by staining for HOXA2. By contrast, DS(DMH-1 and SB431542) treatment-derived neuroepithelial cells showedrostral identity as demonstrated by staining for OTX2.

The efficiency of motor neuron generation from these two populations ofneuroepithelial cells was then compared. After treatment with 0.1 μMRetinoic Acid (RA) and 1 μM purmorphamine (a small molecule foractivating SHH signaling) for another 6 days, more than 90% OLIG2⁺ MNprogenitors were induced from CDS treatment-derived neuroepithelialcells, but only 60% from DS treatment-derived neuroepithelial cells.These data suggest an efficient approach for inducing MN progenitorsfrom pluripotent stem cells by contacting the stem cells with athree-molecule cocktail of CDS (CHIR99021, DMH-1, and SB431542) oranother cocktail of compounds affecting the Wnt pathway, the BMPpathway, and the TGFβ signaling pathway, respectively, as describedherein.

Since the pMN domain is patterned by a gradient of SHH signaling, theefficiency of MN generation upon exposure to different concentrations ofpurmorphamine was examined. (pMN is a special name in neural developmentand represents a progenitor domain for motor neuron specification in thespinal cord.) It was observed that 0.5 μM purmorphamine induced asimilarly pure population of OLIG2⁺ MN progenitors as 1 μM(approximately 90% OLIG2⁺ MN progenitors), but induced few NKX2.2⁺ p3progenitors (V3 interneuron progenitors). Concentrations of less than0.5 μM purmorphamine induced the fewest number of OLIG2⁺ MN progenitors.

Example 2—Long-Term Expansion of OLIG2⁻ MN Progenitors

Next, we examined whether OLIG2⁺ MN precursors could be maintained as acontinuously dividing population. OLIG2⁺ MN precursors obtained from the2-week differentiation were split and cultured under CDS conditions(i.e., in the presence of the 3-molecule CDS cocktail) plus 0.1 μM RAand 0.5 μM purmorphamine. However, the cells gradually lost theirdividing potential and became post-mitotic MNs as determined by stainingfor MNX1, which suggested that RA induces the exit of cell cycle andpromotes neurogenesis. Withdrawing RA from the culture was attempted. Inthe presence of the CDS cocktail plus 0.5 μM purmorphamine, the cellsexpanded but the neural precursors gradually lost OLIG2 expression andincreased NKX2.2 expression, which suggested that purmorphamine alonecannot maintain MN precursors. Instead, the cells switch into p3 domainprecursors. Next, motor neuron progenitors were cultured with the CDScocktail plus RA, purmorphamine, and plus 0.5 mM VPA. VPA can activateNotch signaling pathway, which blocks the neurogenesis induced by RA.This condition can maintain a substantially pure population of OLIG2⁺ MNprogenitors without inducing MNX1⁺ MNs (e.g., a population substantiallydevoid of MNX1⁺ motor neurons) and switch into p3 domain precursors.

Among the four small molecules of the “maintaining” culture medium,CHIR99021 was the core factor for the expansion of MN progenitors sincewithdrawal of CHIR99021 resulted in a significant loss of dividingpotential. DMH-1 and SB431542 cooperated with CHIR99021 to obtain themaximal proliferation. VPA repressed the neurogenesis by blocking theexpression of neurogenic transcription factors Ngn2 and Ngn1. RA andpurmorphamine are required to maintain the expression of MN progenitormarker OLIG2, which means maintaining the identity and differentiationpotential of MN progenitors. Under these conditions, OLIG2⁺ MNprogenitors can be maintained and expanded in culture for at least 5weeks (e.g., at least 5 passages), yielding previously unobtainablenumbers of MN progenitors (on the order of producing 10⁴ MN progenitorsfrom a single MN progenitor cell). It was also observed that OLIG2⁺ MNprecursors can be frozen in liquid nitrogen. When thawed and cultured inMN differentiation medium, OLIG2⁺ MN progenitors differentiated intoMNX1⁺ post-mitotic motor neurons in 1 week and further into CHAT⁻ maturemotor neurons in 2-3 weeks.

Example 3—Differentiating and Maintaining Hindbrain Serotonergic NeuralProgenitors

Human embryonic stem cells or induced pluripotent stem cells were seededonto laminin-coated plates and cultured in human ESC medium for 1 day.(See below for medium components.) On the following day, the culturemedium was changed to Neurobasal culture medium comprising 2 μMSB431542, 2 μM DMH1, and 1.0-3.0 μM CHIR99021 for one week. Neuralprogenitors having hindbrain identity were generated from humanpluripotent stem cells. The hindbrain neural progenitors were defined bytheir expression of hindbrain makers (e.g., GBX2, KROX20, HOXA1-4,HOXB1-4), but not forebrain markers (e.g., FOXG1, OTX2, EMX1, NKX2.1,SIX3), midbrain markers (e.g., EN1, LMX1A, LMX1B, SIM1, LIM1), or spinalcord markers (e.g., HOXB6, HOXB8). The progenitors also included theneural progenitor markers (e.g., SOX1, SOX2, NESTIN, N-Cadherin, andKi67).

To differentiate neural progenitors toward the serotonergic neural cellfate, hindbrain neural progenitors were cultured in a medium comprising1000 ng/mL C25II Sonic Hedgehog (SHH) or 1 μM purmorphamine for oneweek. The resultant cells became ventral hindbrain progenitorsexpressing hindbrain makers (e.g., GBX2, KROX20, HOXA1-4, HOXB1-4), butnot forebrain markers (e.g., FOXG1, OTX2, EMX1, NKX2.1, SIX3). Theresultant cells also expressed ventral hindbrain markers OLIG2, NKX6.1,and NKX2.2. The percentage of NKX2.2⁺ cells was as high as 91% of totalcells assessed using a FACS assay. These ventral hindbrain neuralprogenitors could be maintained in a maintenance culture mediumcomprising 3.0 μM CHIR99021 and 1000 ng/mL C25II Sonic Hedgehog (SHH) or1 μM purmorphamine for at least 5 passages. The ventral hindbrain neuralprogenitors were seeded onto polyornithine-coated coverslips,laminin-coated coverslips, or laminin-coated plates for furtherdifferentiation in a neural differentiation medium comprising 2.5 μMDAPT (a γ-secretase inhibitor and indirect inhibitor of Notch, aγ-secretase substrate) to enhance maturation.

Example 4—Materials and Experimental Procedures

Human ESC lines H9 and H1 (WiCell Institute, NIH Code 0062 and 0043,passages 18-35) and human iPSC lines (iSMA13 and iSMA23) were culturedon irradiated mouse embryonic fibroblasts (MEFs) as described in thestandard hESC protocol available at wicell.org on the World Wide Web.

Retinoic acid, purmorphamine, and SHH stock solutions for addition to aculture medium described herein can be prepared as described by Hu andZhang (Methods Mol. Biol. 636:123-137, 2010).

Generation of OLIG2⁺ MN Progenitors Using a Monolayer DifferentiationMethod:

After treating with 1 mg/ml Dispase, hPSCs were split 1:6 on irradiatedMEFs. On the following day, the culture medium was replaced with neuralmedium (DMEM/F12, Neurobasal® culture medium (Life Technologies) at 1:1,1× N2 neural supplement, 1× B27 neural supplement, 1 mM ascorbic acid).3 μM CHIR99021, 2 μM DMH-1, and 2 μM SB431542 were added in freshmedium. The culture medium was changed daily. Human PSCs maintainedunder these conditions for 6 days were induced into neuroepithelialcells. When treated with 1 mg/ml Dispase, neuroepithelial cells weresplit at 1:6 on irradiated MEF with the same medium described above. 0.1μM RA and 0.5 μM purmorphamine were added in combination with CHIR99021,DMH-1, and SB431542. The medium was changed daily. Neuroepithelial cellsmaintained under these conditions for 6 days differentiated into OLIG2⁺MN progenitors. We note that this method worked with and without MEFs.

Generation of OLIG2⁺ MN Progenitors Using a Suspension DifferentiationMethod:

After treating with 1 mg/ml Dispase, hPSCs were lifted and cultured ascell aggregates in suspension in hESC medium (DMEM/F12 medium+20%KnockOut™ Serum Replacement (Gibco) supplement, 1× NMAA, 1× glutamax)for four days. On day 4, the hESC medium was replaced with neural medium(DMEM/F12, Neurobasal® culture medium (Life Technologies) at 1:1, 1× N2neural supplement, 1× B27 neural supplement, 1 mM ascorbic acid). Afterculturing for another two days, the cell aggregates were attached on theculture plate. The neural medium was changed every other day. Afterculturing under these conditions for one week, hPSCs were induced intoneuroepithelial cells. After treating with 1 mg/ml Dispase,neuroepithelial cells were lifted again and cultured as neurospheres insuspension. 0.1 μM RA and 0.5 μM purmorphamine were added in neuralmedium. The medium was changed every other day. Neuroepithelial cellsmaintained under these conditions for ten days differentiated intoOLIG2+MN progenitors.

Maintenance of OLIG2⁺ MN Progenitors:

OLIG2⁺ MN progenitors can be frozen in regular freezing medium(DMEM/F12, 10% fetal bovine serum, 10% DMSO). To passage, MN progenitorswere treated with 1 mg/ml Dispase and split 1:6 on irradiated MEFs.CHIR99021, DMH-1, SB431542, VPA, purmorphamine, and RA were added atsame concentrations as described above. To induce differentiation intomature MNs, CHIR99021, DMH-1, and SB431542 were withdrawn from themedium, and MN progenitors were cultured in the basic neural medium(DMEM/F12, Neurobasal medium at 1:1, 1× N2 neural supplement, 1× B27neural supplement, and 1 mM ascorbic acid) plus 0.1 μM RA and 0.1 μMpurmorphamine for 1 week to generate MNX1⁺ post-mitotic MNs, and thendifferentiated into CHAP mature MNs in another 1-2 weeks.

Example 5—Generation and Expansion of Pure Motor Neuron Progenitors fromHuman Stem Cells

Summary

Human pluripotent stem cells (hPSCs) have opened new opportunities forunderstanding early human development, modeling disease processes anddeveloping new therapeutics. However, these applications are hindered bylow efficiency and heterogeneity of target cell types differentiatedfrom hPSCs, such as motor neurons (MNs), as well as our inability tomaintain the potency of lineage committed progenitors. By using acombination of small molecules that regulate multiple signalingpathways, we developed a novel method to guide hPSCs to a near-purepopulation (>95%) of OLIG2⁺ motor neuron progenitors (MNPs) in 12 days,and a highly enriched population (>90%) of functionally mature MNs inanother 16 days. More importantly, the OLIG2⁺ MNPs can be expanded forat least 5 passages so that a single MNP can be amplified to 1×10⁴. TheMNs produced from the expandable MNPs exhibit functional properties,including formation of neuromuscular junctions when co-cultured withskeletal muscle cells and projection of axons toward muscles whengrafted into the developing chick spinal cord. The consistent and highlyenriched MN populations enable modeling MN degenerative diseases anddeveloping large-scale, MN based screening assays.

Introduction

Human pluripotent stem cells (PSCs), including embryonic stem cells(ESCs) and induced pluripotent stem cells (iPSCs), offer a new modelsystem to explore early human development and dissect disease processes,as well as an opportunity to devise therapeutics (Grskovic, M.,Javaherian, A., Strulovici, B. & Daley, G. Q. Induced pluripotent stemcells—opportunities for disease modeling and drug discovery. Nat RevDrug Discov Vol. 10, 915-929, 2011; Han, S. S., Williams, L. A. & Eggan,K. C. Constructing and deconstructing stem cell models of neurologicaldisease. Neuron Vol. 70, 626-644, 2011; Goldman, S. A., Nedergaard, M. &Windrem, M. S. Glial progenitor cell-based treatment and modeling ofneurological disease. Science Vol. 338, 491-495, 2012). A criticalrequirement for achieving these potentials is directed differentiationof hPSCs to target cell types. Substantial progress has been made inguiding hPSCs to major cell lineages, including blood, cardiac, andneural cells (Ma, F. et al. Generation of functional erythrocytes fromhuman embryonic stem cell-derived definitive hematopoiesis. Proc NatlAcad Sci USA Vol. 105, 13087-13092, 2008; Kattman, S. J. et al.Stage-specific optimization of activin/nodal and BMP signaling promotescardiac differentiation of mouse and human pluripotent stem cell lines.Cell Stem Cell Vol. 8, 228-240, 2011; Liu, H. & Zhang, S. C.Specification of neuronal and glial subtypes from human pluripotent stemcells. Cell Mol Life Sci Vol. 68, 3995-4008, 2011). Nevertheless,generation of pure or highly enriched cells, which are often necessaryfor biochemical analysis, disease modeling, and clinical application,has not been readily achieved. In particular, it is often necessary toinduce hPSCs to functionally specialized subtypes of cells, which areonly a tiny fraction of cells in a normal tissue/organ of our body. Sucha need poses critical challenges to the stem cell field.

Spinal motor neurons (MNs) are a highly specialized type of neurons thatreside in the ventral horns and project axons to muscles to controltheir movement. Degeneration of MNs is implicated in a number ofdevastating diseases, including spinal muscular atrophy (SMA),amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth (CMT) andpoliomyelitis (polio) disease. The above disease iPSCs have beengenerated from patients and attempts have been made to identifydisease-related phenotypes and to dissect out the underlying mechanismsbefore embarking on drug discovery (Ebert, A. D. et al. Inducedpluripotent stem cells from a spinal muscular atrophy patient. NatureVol. 457, 277-280, 2009; Egawa, N. et al. Drug screening for ALS usingpatient-specific induced pluripotent stem cells. Sci Transl Med Vol. 4,145ra104, 2012; Chen, H. et al. Modeling ALS with iPSCs reveals thatmutant SOD1 misregulates neurofilament balance in motor neurons. CellStem Cell Vol. 14, 796-809, 2014; Kiskinis, E. et al. Pathways disruptedin human ALS motor neurons identified through genetic correction ofmutant SOD1. Cell Stem Cell Vol. 14, 781-795, 2014). However, theseefforts are hindered by our inability to produce pure or highly enrichedMNs with consistent quality. A number of protocols have been developed,including neural progenitor induction followed by neural patterning byretinoic acid (RA) and sonic hedgehog (SHH) (Li, X. J. et al.Specification of motoneurons from human embryonic stem cells. NatBiotechnol Vol. 23, 215-221, 2005; Qu, Q. et al. High-efficiency motorneuron differentiation from human pluripotent stem cells and thefunction of Islet-1. Nat Commun Vol. 5, 3449, 2014), neural progenitorinduction followed by genetic manipulations using adenovirus-mediatedgene delivery (Hester, M. E. et al. Rapid and efficient generation offunctional motor neurons from human pluripotent stem cells using genedelivered transcription factor codes. Mol Ther Vol. 19, 1905-1912,2011), and differentiation of MNs with above methods followed by sortingwith GFP labeling under MNX1 (also known as HB9) promoter (Amoroso, M.W. et al. Accelerated high-yield generation of limb-innervating motorneurons from human stem cells. J Neurosci Vol. 33, 574-586, 2013). Thesedifferentiation protocols are tedious, time consuming (1 to 2 months),and are of low efficiencies (30-70%) unless by sorting (90%).Furthermore, PSC-derived neurons in vitro, including MNs, are oftenimmature, making it difficult to reveal disease phenotypes that aremanifested in functionally mature cells. Hence, there is a critical needto develop a new method that would enable generation of pure andfunctionally mature MNs with consistent quality and in a short time.

By using a combination of small molecules in a chemically-defined neuralmedium, we have guided hPSCs to a near-pure population of OLIG2⁺ motorneuron progenitors (MNPs) in 12 days, and a highly enriched populationof functionally mature MNs (>90%) in another 16 days by the use of aNotch inhibitor. Furthermore, we developed a new method to expand asingle MNP to 1×10⁴ MNPs, enabling production of a large quantity(5×10⁵) of consistent MNs from a single stem cell. (We expand a singleMNP to approx. 10,000 fold and a single PSC generates approx. 50 MNPsduring the first two steps. Therefore, we generate approx. 10,000 MNPsfrom an MNP and 500,000 MNPs from a PSC.) MNs generated by the novelmethod display molecular phenotypes of SMA and ALS, and can be readilyadapted to screening platforms, as illustrated by our luciferase-basedaxonal length assay using ALS patient MNs.

Results

1. Generation of Pure MNPs by a Small-Molecule Cocktail

Specification of MNPs follows serial and overlapping developmentalsteps: neural induction as well as caudalization and ventralization ofneuroepithelial progenitors (NEPs) (Li, X. J. et al. Specification ofmotoneurons from human embryonic stem cells. Nat Biotechnol Vol. 23,215-221, 2005; Wichterle, H., Lieberam, I., Porter, J. A. & Jessell, T.M., Directed differentiation of embryonic stem cells into motor neurons.Cell Vol. 110, 385-397, 2002). In the presence of small molecules,SB431542 (inhibitor of activin-nodal signaling, 2 μM) and DMH1(inhibitor of BMP signaling, 2 μM) (Chambers, S. M. et al. Highlyefficient neural conversion of human ES and iPS cells by dual inhibitionof SMAD signaling. Nat Biotechnol Vol. 27, 275-280, 2009; Neely, M. D.et al. DMH1, a highly selective small molecule BMP inhibitor promotesneurogenesis of hiPSCs: comparison of PAX6 and SOX1 expression duringneural induction. ACS Chem Neurosci Vol. 3, 482-491, 2012), hESCs (lineH9) were induced to NEPs, with 77±9% of the total differentiated cellsbeing SOX1⁺ (FIG. 4A, B). Activation of WNT by a small molecular agonistCHIR99021 has been shown to promote neural induction and potentiallyalso neuroepithelial proliferation (Li, W. et al. Rapid induction andlong-term self-renewal of primitive neural precursors from humanembryonic stem cells by small molecule inhibitors. Proc Natl Acad SciUSA Vol. 108, 8299-8304, 2011; Lu, J. et al. Generation ofintegration-free and region-specific neural progenitors from primatefibroblasts. Cell Rep., Vol. 3, 1580-1591, 2013). Under the treatment of3 μM CHIR together with 2 μM SB+2 μM DMH-1 for 6 days, nearly all thedifferentiated cells were SOX1⁺ NEPs (>98%) (FIG. 4A, B). CHIR not onlyaugmented the efficiency of neural induction, but also increased theyield of SOX1⁺ NEPs by 2.8 fold (FIG. 4B). Wnt activation (by CHIR)often induces a caudal fate of neural progenitors (Xi, J. et al.Specification of midbrain dopamine neurons from primate pluripotent stemcells. Stem Cells Vol. 30, 1655-1663, 2012). Indeed, CHIR-induced NEPsshowed a caudal identity with HOXA3 expression. In contrast, NEPsinduced by SB+DMH1 (without CHIR) exhibited a rostral identity with OTX2expression (FIG. 4B). Therefore, treatment of CHIR+SB+DMH-1 combines thesteps of induction and caudalization of NEPs, representing achemically-defined, single-step method for obtaining homogenous caudalNEPs from hPSCs.

The next step is to specify OLIG2⁺ MNPs by mimicking the ventralizationof NEPs in vivo. By exposing the CHIR+SB+DMH-1-induced caudal-like NEPsto RA (0.1 μM) and Pur (Purmorphamine, SHH signalling agonist, 1 μM) for6 days, which was identified in our previous study (Li, X. J. et al.Directed differentiation of ventral spinal progenitors and motor neuronsfrom human embryonic stem cells by small molecules. Stem Cells Vol. 26,886-893, 2008), we obtained 81±9% OLIG2-expressing neural progenitors(FIG. 4C). However, about 40% OLIG2⁺ cells co-expressed NKX2.2, anotherventral spinal cord marker (FIG. 4C). During neural development, OLIG2and NKX2.2 are initially induced in a common pool of progenitors thatultimately segregate into unique territories giving rise to distinctOLIG2⁺ MNPs and NKX2.2⁺ interneuron progenitors (Lee, S. K. & Pfaff, S.L. Transcriptional networks regulating neuronal identity in thedeveloping spinal cord. Nat Neurosci., Vol. 4 Suppl, 1183-1191, 2001).WNT signalling plays a critical role in this segregation (Wang, H., Lei,Q., Oosterveen, T., Ericson, J. & Matise, M. P. Tcf/Lef repressorsdifferentially regulate Shh-Gli target gene activation thresholds togenerate progenitor patterning in the developing CNS. Development Vol.138, 3711-3721, 2011). We thus added WNT agonist CHIR in combinationwith RA and Pur. Under the treatment of CHIR+RA+Pur for 6 days, NKX2.2expression was completely repressed in the culture, but OLIG2-expressingpopulation was also decreased to 62±5% (FIG. 4C). We reasoned that WNTsignalling may elevate the threshold of SHH signalling necessary toinduce OLIG2 expression. However, at the increased concentration, SHHagonist Pur became toxic to the NEPs. We thus took an alternativeapproach to decrease the threshold of SHH signalling by repressing thedorsalizing molecule of the spinal cord, BMP signalling. Addition ofdual SMAD inhibitors SB and DMH-1 in combination with CHIR+RA+Pursignificantly increased OLIG2⁺/NKX2.2⁻ cell population (FIG. 4C). Byserial titration of Pur and CHIR in combination with RA, SB and DMH-1(FIG. 8), we found that treatment with 1 μM CHIR, 2 μM SB, 2 μM DMH-1,0.1 μM RA and 0.5 μM Pur for 6 days resulted in a robust population ofOLIG2⁺ MNPs (95±3%), among which few (<0.5%) OLIG2/NKX2.2 doublepositive cells were found (FIG. 4C).

Our protocol for OLIG2+MNP specification is highly reproducible inmultiple different hPSC lines, including normal iPSC line IMR90, ALSiPSC lines SOD1-D90A and SOD1-A4V, and SMA iPSC lines SMA13 and SMA232.Under the treatment of CHIR+SB+DMH1 for 6 days and CHIR+SB+DMH1+RA+Purfor another 6 days, all the hPSC lines generated more than 90% OLIG2⁺MNPs (FIG. 4D). Thus, coordinated specification and patterning ofneuroepithelia by small molecules lead to robust generation of purepopulations of region-specific MNPs.

2. MNPs are Expanded to Large Numbers.

Developmentally, OLIG2⁺ MNPs are present transiently and they transitionto other neuronal (interneuron) and glial (oligodendrocyte) lineagesafter generation of MNs. For cellular and biochemical analysis, it iscrucial to expand the MNPs without losing their ability to produce MNs,which has not been achieved. Since the CHIR+SB+DMH1+RA+Pur condition ishighly efficient in specifying and generating MNPs, we asked if thecondition could expand the OLIG2⁺ MNPs in a continuously dividing state.We first examined whether RA and Pur are required for maintaining OLIG2expression. The MNPs were passaged weekly under the CHIR+SB+DMH1condition with or without Pur or RA+Pur. After two passages, OLIG2⁺ MNPswere decreased to 35±5% in the control group (without Pur and RA), to62±5% in Pur group, and a large population of NKX2.2⁺ cells appeared inthese two groups. In the RA+Pur group, OLIG2⁺ MNPs were maintained at91±3% with rare NKX2.2⁺ cells (FIG. 5A). Therefore, RA and Pur arerequired for maintaining the identity of MNPs.

We then examined whether CHIR, SB and DMH-1 are required for maintainingcell proliferation. The MNPs were passaged under the RA+Pur conditionand divided into three groups: SB+DMH-1 treatment group, CHIR treatmentgroup, and CHIR+SB+DMH-1 treatment group. After two passages, the cellpopulation expressing Ki-67, a cell proliferation marker, was at lessthan 8%, 81±6%, and 92±4% in the SB+DMH-1 group, the CHIR group, and theCHIR+SB+DMH-1 group, respectively (FIG. 5B). Therefore, CHIR+SB+DMH-1are required for maintaining the proliferation of MNPs at the maximumlevel.

When the MNPs were expanded in the same medium (CHIR+SB+DMH-1+RA+Pur)for longer than two passages, the OLIG2-expressing MNP populationdecreased with a concomitant appearance of MNX1 expressing MNs,suggesting that some MNPs have exited cell cycle and differentiated toneurons. We reasoned that this is likely due to the neurogenic effect ofRA. It was known that valproic acid (VPA), a histone deacetylaseinhibitor, can repress neurogenesis by indirectly activating Notchsignalling (Stockhausen, M. T., Sjölund, J., Manetopoulos, C. & Axelson,H. Effects of the histone deacetylase inhibitor valproic acid on Notchsignalling in human neuroblastoma cells. Br J Cancer Vol. 92, 751-759,2005). We thus added VPA to the culture system. Under this culturecondition, the MNPs were expanded for at least 5 passages yet maintainedOLIG2 expression at 82±9% (FIG. 5D). Further culturing under thiscondition resulted in gradual decrease of OLIG2⁺ cell population andincrease of NKX2.2±cell population, suggesting a need of an alternativestrategy for an even longer term expansion. Nevertheless, continualexpansion of MNPs for 5 passages allows amplification of a single MNP to1×10⁴ MNPs, translating to the generation of >5×10⁵ MNPs from a singlehPSC (FIG. 5E). Furthermore, these MNPs can be frozen and thawed inregular conditions with over 90% recovery.

3. MNPs Differentiate into Enriched Functional MNs.

To determine the differentiation of expanded MNPs, we withdrewCHIR+SB+DMH-1, increased RA concentration (0.5 μM), and reduced Pur (0.1μM). After 6 days, nearly all the MNPs differentiated into MNs, asevidenced by expression of MNX1 (90±9%) or ISL1 (95±3%) (FIG. 6A, B).Further culture on MATRIGEL or astrocyte feeders for two weeks resultedin generation of more mature MNs that expressed CHAT, although the CHAT⁺MN population (47±9%) was substantially lower than the MNX1⁺ MNPs. Wereasoned that the lower population of CHAT⁺ mature MNs may be due toproliferation of the small number of neural precursors and theirsubsequent differentiation to other neuronal types via lateralinhibition of NOTCH signaling (Lewis, J., Notch signalling and thecontrol of cell fate choices in vertebrates. Semin Cell Dev Biol Vol. 9,583-589, 1998). To overcome this inefficiency of MN maturation, weapplied Compound E (Cpd E), a NOTCH signaling inhibitor in the MNculture. CpdE treatment resulted in a near homogenous MAP2⁺ matureneuronal cultures without any proliferating cells (Ki67⁺), and about91±6% of MAP2⁺ neurons expressed CHAT (FIG. 6A, B). These CHAT+MNs wereelectrophysiologically active, as defined by their ability to elicitaction potentials in response to depolarizing current injection incurrent-clamp recordings (FIG. 9). Therefore, CpdE not only increasesthe mature MN population but also substantially shortens the maturationprocess.

To determine whether the CpdE-accelerated MNs are functional, weco-cultured the MNs with differentiated myotubes from mouse C2C12 cells.After 10 days of co-culture, we observed aggregated BTX⁺ acetylcholinereceptors on myotubes and their overlapping with CHAT⁺ neurites (FIG.6C), suggesting formation of neuromuscular junctions. To study theability of motor neurons to project axons toward the muscle targets,CpdE treated MNs were transplanted in ovo into the lesioned neural tubeof chicken embryos at HH stage 15-16. Transplanted embryos showedsuccessful engraftment of human MNs (as marked by GFP expression) intothe ventral horn (FIG. 6D). Importantly, we observed GFP labelled humanMN axons (CHAT⁺) projected ventrally through the ventral roots and alongthe peripheral nerves of the host (FIG. 6D′). These data indicate thatmature MNs generated by CpdE treatment exhibit proper functions.

4. Enriched MNs Enable Presentation of Disease Phenotypes and Buildingof Screening Platforms.

Most neurodegenerative diseases, like SMA and ALS, preferentially affectone type of neurons such as MNs. Genetically linked disorders, includingSMA and ALS, may lead to changes in gene dosage of less than 50%. Hence,it will be technically difficult to discern changes in gene expressionif the population of disease target cells is not highly enriched. Todetermine the utility of the MNs generated with the above method, wemeasured the expression of genes that are known to be altered in SMA andALS. In this analysis, we generated spinal non-MNs from the same iPSCsas a control by replacing Pur with Cyclopamine (Cyc) to block SHHsignaling (FIG. 10A). Under RA and Cyc treatment for 6 days, the inducedspinal neural precursors were void of OLIG2 expression, and thedifferentiated neurons were void of MNX1 and CHAT expression, but withGABA expression (FIG. 10B, C). Using these highly enriched MNs and GABAneurons, we found that the mRNA of survival motor neuron (SMN, theprotein affected in SMA) was decreased in both MNs and GABA neurons thatwere derived from SMA patients as compared to those from non-SMA iPSCs(FIG. 7A). This is consistent with the fact that SMN mutations affectall cell types. Interestingly, we found that MNs exhibited even lower(38±4%) SMN than GABA neurons (60±6%) (FIG. 7A), again consistent with aprevious report that MNs express markedly lower levels of full-lengthSMN transcripts from SMN2 gene than do other cells in the spinal cord(Ruggiu, M. et al. A role for SMN exon 7 splicing in the selectivevulnerability of motor neurons in spinal muscular atrophy. Mol Cell Biol32, 126-138, 2012). Similarly in ALS caused by mutations in superoxidedismutase (SOD1) gene, MNs display neurofilament (NF) aggregation thatis attributed to the decreased level of light polypeptide neurofilament(NEFL) (Chen, H. et al. Modeling ALS with iPSCs reveals that mutant SOD1misregulates neurofilament balance in motor neurons. Cell Stem Cell 14,796-809, 2014). We found a 45±4% reduction of NEFL mRNA in D90A MNs, butnot GABA neurons, when compared to genetically corrected (D90D) MNs andGABA neurons (FIG. 7B). Together, these data indicate that the enrichedMNs generated from patient iPSCs using our new method enableidentification of disease related phenotypes.

Our ability to generate large quantities of consistent MNs offers anopportunity for building high-throughput screening platforms for MNdiseases. In ALS, astrocytes enhance disease progression by promotingaxonal degeneration and MN death (Di Giorgio, F. P., Carrasco, M. A.,Siao, M. C., Maniatis, T. & Eggan, K. Non-cell autonomous effect of gliaon motor neurons in an embryonic stem cell-based ALS model. Nat NeurosciVol. 10, 608-614, 2007; Nagai, M. et al. Astrocytes expressingALS-linked mutated SOD1 release factors selectively toxic to motorneurons. Nat Neurosci Vol. 10, 615-622, 2007; Haidet-Phillips, A. M. etal. Astrocytes from familial and sporadic ALS patients are toxic tomotor neurons. Nat Biotechnol Vol. 29, 824-828, 2011). Indeed, when ALSiPSC-derived MNs were grown on top of ALS (D90A SOD1) or geneticallycorrected (D90D SOD1) astrocytes (FIG. 11) in a medium that lacksneurotrophic factors, MNs began to show neurite fragmentation withreduced neurite length on D90A but not D90D astrocytes at Day 10 (FIG.7C). To enable automated measurement of neurite length for highthroughput screening, we established a reporter iPSC line (from D90ASOD1) with a luciferase reporter NanoLuc (Nluc) fused with SYNAPTOPHYSIN(SYP) (FIG. 7D), a synaptic vesicle glycoprotein, which targets the Nlucreporter to axonal membrane, not cytoplasm (Nakata, T., Terada, S. &Hirokawa, N. Visualization of the dynamics of synaptic vesicle andplasma membrane proteins in living axons. J Cell Biol Vol. 140, 659-674,1998). We first established the linear relationship between Nlucexpression and MN numbers by measuring luciferase activity of cultures10 days after plating different numbers of SYP-Nluc expressing MNs(1250, 2500, 5000, 10000 and 20000 cells) on astrocytes (FIG. 12A). Wethen tested whether the Nluc reporter activity is correlated with thereduced axonal length. Same numbers of SYP-Nluc expressing MNs wereplated on D90D and D90A astrocytes and the Nluc activity was detected atDay 10. The Nluc activity on D90A astrocytes was significantly decreasedto 70.5±2.7%, comparing to the D90D astrocyte group (FIG. 12B). Next, weexposed the co-cultures to three compounds, Riluzole (Rilu) (Miller, R.G., Mitchell, J. D. & Moore, D. H. Riluzole for amyotrophic lateralsclerosis (ALS)/motor neuron disease (MND). Cochrane Database Syst RevVol. 3, CD001447, 2012), the only approved drug for ALS, as well asKenpaullone (Ken) and EphA inhibitor (EphAi) that are known to rescueaxonal degeneration in ALS cell models (Yang, Y. M. et al. A smallmolecule screen in stem-cell-derived motor neurons identifies a kinaseinhibitor as a candidate therapeutic for ALS. Cell Stem Cell Vol. 12,713-726, 2013; Van Hoecke, A. et al. EPHA4 is a disease modifier ofamyotrophic lateral sclerosis in animal models and in humans. Nat Med.,Vol. 18, 1418-1422, 2012). Riluzole had no effect on Nluc activity. Kenand EphAi increased the Nluc activity to 1.5 and 2.8 fold in both D90Aand D90D groups (FIG. 7E), indicating that they increased axonal length.However, Ken and EphAi did not specifically rescue the axonal length ofMNs induced by ALS astrocytes, as the ratio of Nluc activity on D90Aastrocytes versus D90D astrocytes remained at 70% (FIG. 7E). Theseresults provide a proof of principle for the use of our enriched,patient-derived MNs for drug screening and suggest its potential foridentifying disease specific targets.

DISCUSSION

We have developed a novel strategy for guiding hPSCs to a near-purepopulation of OLIG2⁺ MNPs in 12 days by coordinating signaling pathwaysusing small molecules, and subsequently a highly enriched population offunctionally mature MNs (>90%) in another 16 days by the use of a Notchinhibitor.

Furthermore, we have devised a method to expand a single MNP to 1×10⁴MNPs, enabling production of a large quantity (5×10⁵) of consistent MNsfrom a single stem cell. Our novel method enables presentation ofdisease phenotypes and building of screening platforms, as illustratedby our luciferase-based axonal length assay using ALS patient MNs.

Compared to previous methods, our method has at least two criticalimprovements. The first is the application of WNT agonist during MNPdifferentiation. WNT signaling is a more efficient pathway to caudalizeneural progenitors (Xi, J. et al. Specification of midbrain dopamineneurons from primate pluripotent stem cells. Stem Cells Vol. 30,1655-1663, 2012). Thus, the combination of WNT activator (CHIR99021)with BMP inhibitor (DMH1) and TGFb inhibitor (SB431542) inducedhomogenous caudal NEPs from hPSCs. Most importantly, WNT signaling playsa critical role in MNP specification. All previous methods used RA andSHH to induce OLIG2⁺ MNPs without examining other ventral spinalmarkers, especially co-expression of NKX2.2. We showed here that use ofRA and SHH generated mixed ventral progenitors with a large populationof cells that co-express OLIG2 and NKX2.2. During spinal corddevelopment, OLIG2 and NKX2.2 are initially induced in a common pool ofprogenitors that ultimately segregate into unique territories, givingrise to distinct OLIG2⁺ MNPs (pMN domain) and NKX2.2⁺ interneuronprogenitors (p3 domain) (Lee, S. K. & Pfaff, S. L. Transcriptionalnetworks regulating neuronal identity in the developing spinal cord. NatNeurosci Vol. 4 Suppl, 1183-1191, 2001). Without segregation, NKX2.2could interfere with the differentiation of OLIG2⁺ progenitors to MNX1⁺MNs, which is one of the reasons why some previous methods induced ahigh percentage of OLIG2⁺ progenitors, but ended with a small populationof MNX1⁺ MNs. WNT signaling was reported to selectively opposeSHH-mediated induction of NKX2.2, but have little effect on OLIG2, andthereby establish their distinct expression domains in cooperation withgraded SHH signaling (Wang, H., Lei, Q., Oosterveen, T., Ericson, J. &Matise, M. P. Tcf/Lef repressors differentially regulate Shh-Gli targetgene activation thresholds to generate progenitor patterning in thedeveloping CNS. Development Vol. 138, 3711-3721, 2011). As WNT elevatesthe strength of SHH signaling to induce OLIG2 expression, the twoinhibitors of dorsalizing BMP signaling (Dorsomorphin and LDN-193189)were also included. Therefore, our method of combining small moleculesregulating WNT, SHH, RA and BMP signals closely mimics the cooperationof these signals in the spinal cord development in vivo to specify theregion-specific OLIG2⁺ MNPs.

The second improvement is the application of a NOTCH inhibitor during MNmaturation. Lateral inhibition mediated by NOTCH signaling is anintrinsic mechanism to guide orderly transition of mitotically activeprecursors into different types of post-mitotic neurons and glia atdifferent stages (Lewis, J. Notch signaling and the control of cell fatechoices in vertebrates. Semin Cell Dev Biol Vol. 9, 583-589, 1998). Thetreatment with NOTCH inhibitor CpdE in our method synchronizes thedifferentiation of OLIG2⁺ MNPs to generate homogenous mature MNs withoutmixing with any other neural cells. With these two improvements, ourmethod robustly generates almost homogenous mature MNs, which exhibitfunctional properties, including formation of neuromuscular junctionswhen co-cultured with skeletal muscle cells and projection of axonstoward muscles when grafted into the developing chick spinal cord. Moresignificantly, the MNs derived from disease iPSCs by our method exhibitthe MN-specific molecular phenotypes, including down-regulation offull-length SMN in SMA and down-regulation of NEFL level in ALS, whichwould be nearly impossible to detect with previous methods that onlygenerate a small population of MNs in the mixed culture.

A large quantity of consistent target cells, such as mature MNs, isnecessary for high-throughput screening. In general, lineage committedprogenitors can be expanded, but quickly lose their differentiationpotency. For example, OLIG2⁺ MNPs can be expanded with FGF and/or EGF,but quickly lose the potency of MN differentiation in two passages.Several recent reports described the expansion of neural progenitorswith small molecules of WNT and/or SHH signaling (Li, W. et al. Rapidinduction and long-term self-renewal of primitive neural precursors fromhuman embryonic stem cells by small molecule inhibitors. Proc Natl AcadSci USA Vol. 108, 8299-8304, 2011; Reinhardt, P. et al. Derivation andexpansion using only small molecules of human neural progenitors forneurodegenerative disease modeling. PLoS One Vol. 8, e59252, 2013).However, as shown in this study (FIG. 5A), their ability to maintain MNpotential is still significantly diminished during cell passagescompared to our method. Our method can expand MNPs for at least 5passages to amplify a single MNP to 1×10⁴ MNPs, or generate 5×10⁵ MNsfrom single hPSC. This provides a sufficient cell source forhigh-throughput drug screening, as shown in our screening platform forMN axonal degeneration. In summary, our new method enables generation oflarge quantities of MNs with consistency and high purity, providing abasis for modeling MN diseases in vitro and for drug discovery.

Methods

Human Pluripotent Stem Cells (PSCs).

The human PSC lines used in this study are listed in Table 1.Fibroblasts from a 50-y-old female ALS patient carrying the D90A SODImutation (ND29149, Coriell Institute, coriell.org), a 3-y-old male SMApatient (GM03813, Coriell Institute) and a 7-m-old SMA patient (GM00232,Coriell Institute) were reprogrammed using the non-integrating Sendaivirus as described (Ban et al., 2011) to established iPSC linesALS-D90A, SMA13 and SMA232. D90D iPSC line was established by correctingthe D90A SODI mutation in ALS-D90A lines by TALEN technology (Chen etal., 2014). A4V SODI mutant ALS iPSC line, established with retrovirus,was obtained from Coriell (ND35671). Human ESC line H9 (W A09 line, NIHregistry 0046) and normal iPSC line IMR90-4 were obtained from WiCell.All the PSCs were cultured on irradiated mouse embryonic fibroblasts(MEFs).

MNP Specification and MN Differentiation.

To generate MNPs, hPSCs were dissociated with Dispase (1 mg/ml) andsplit 1:6 on irradiated MEFs or MATRIGEL™ coated plates. On thefollowing day, the PSC medium was replaced with a chemically definedneural medium, including DMEM/F12, Neurobasal medium at 1:1, 0.5×N2,0.5×B27, 0.1 mM ascorbic acid (Santa Cruz), 1×Glutamax and1×penicillin/streptomycin (All other reagents, such as culture mediumminus absorbic acid, from Invitrogen). CHIR99021 (3 uM, Torcris), 2 μM(Tocris) and 2 μM SB431542 (Stemgent) were added in the medium. Theculture medium was changed every other day. Human PSCs maintained underthis condition for 6 days were induced into NEP cells. The NEP cellswere then dissociated with Dispase (1 mg/ml) and split at 1:6 with thesame medium described above. RA (0.1 μM, Stemgent) and 0.5 μMPurmorphamine (Stemgent) were added in combination with 1 μM CHIR99021,2 μM and 2 μM SB431542. The medium was changed every other day. NEPcells maintained under this condition for 6 days differentiated intoOLIG2+ MNPs. The OLIG2⁺ MNPs were expanded with the same mediumcontaining 3 μM CHIR99021, 2 μM DMH-1, 2 μM SB431542, 0.1 μM RA, 0.5 μMPurmorphamine and 0.5 mM VPA (Stemgent), and split 1:6 to 1:8 once aweek with Dispase (1 mg/ml). OLIG2⁺ MNPs were frozen with the regularfreezing medium (DMEM/F12, 10% fetal bovine serum and 10% DMSO) inliquid nitrogen, and cultured again in expansion medium after thawing.

To induce MN differentiation, OLIG2⁺ MNPs were dissociated with Dispase(1 mg/ml) and cultured in suspension in the above neural medium with 0.5μM RA and 0.1 μM Purmorphamine. The medium was changed every other day.OLIG2⁺ MNPs under this condition for 6 days differentiated into MNX1⁺MNs. The MNX1⁺ MNs were then dissociated with Accumax™ (eBioscience)into single cells and plated on MATRIGEL™ coated plates or onastrocytes. The MNX1⁺ MNs were cultured with 0.5 μM RA, 0.1 μMPurmorphamine and 0.1 μM Compound E (Calbiochem) for 10 days to matureinto CHAT⁺ MNs. Insulin-like growth factor 1(IGF-1), brain-derivedneurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF) (allfrom R&D, 10 ng/ml each) were added if MNs were plated at low density(for example, at about 10,000 cells per cm² the MNs grow as a singleneuron, not a cluster). For identifying MN disease phenotypes, SMA andALS MNs were cultured without these factors.

Functional Analysis of Mature MNs.

Whole-cell patch-clamp recordings were performed on iPSC-derived CHAT⁺neurons at Day 28 after iPSC differentiation as we described (Chen, H.et al. Modeling ALS with iPSCs reveals that mutant SOD1 mis-regulatesneurofilament balance in motor neurons. Cell Stem Cell Vol. 14, 796-809,2014).

To examine neuromuscular junction formation, C2C12 cells were seeded onMATRIGEL coated plate in DMEM with 10% FBS, and then were induced toform myotube by switching to DMEM containing 2% FBS. Day 18 MNX1⁺ MNsderived from hPSCs were plated onto myotubes and cultured in maturationcondition for 7 days, after which the neuromuscular synapses werevisualized using CHAT and BTX staining.

To perform transplantations, Day-18 MNX1⁺ MN spheres were trituratedwith a 1-ml pipette tip 5-7 times and treated with CpdE for 24 hrs. (Wemostly culture the cells as adherent cultures in our methods. In atransplantation application we use spheres because the detached anddissociated MNs don't survive well in transplantation.) Transplantationwas performed as previously described (Wichterle, H., Lieberam, I.,Porter, J. A. & Jessell, T. M. Directed differentiation of embryonicstem cells into motor neurons. Cell Vol. 110, 385-397, 2002). Briefly,after a small suction lesion at the prospective intraspinal site wascreated in a chick embryo at stage 15-18 at somites 15-20, MN sphereswere loaded into a handheld micro-injector and placed into the lesion.After 6 days, the chicks were sacrificed, fixed with 4% PFA for 2 h at4° C., and neurite outgrowth was accessed by cutting 30 μm sections ofthe spinal cord near the injected site.

qPCR Analysis

Total RNAs were isolated with RNeasy Plus Mini Kit (Qiagen) according tothe manufacturer's instructions. For qPCR, cDNA was synthesized from 1μg total RNAs using iScript™ reverse transcription supermix (Biorad).qPCR was performed using iTaq™ Universal SYBR® Green Supermix (Biorad).Sequences of the primers are shown in Table 2. GAPDH gene was used asinternal control to equalize cDNA.

Immunostaining and Microscopy

Immunohistochemical staining was performed according to Zhang et al(2001). The following primary antibodies were used: SOX1 (gIgG 1:1000,R&D), OTX2 (mIgG, 1:2000, DSHB), HOXA3 (mIgG 1:1000, R&D), OLIG2 (rIgG1:500, Chemicon), NKX2.2 (mIgG 1:100, DSHB), Ki67 (rIgG 1:200,Chemicon), MNX1 (mIgG 1:50, DSHB), ISL1 (mIgG 1:1000, DSHB), TUJ1 (rIgG1:5000, Covance), CHAT (gIgG 1:300, Chemicon), MAP2 (mIgG 1:1000,Chemicon), GABA (mIgG 1:1000, Chemicon), FoxP1 (rIgG, 1:1000, Chemicon).

MN-Astrocyte Co-Culture and Luciferase Assay

The luciferase reporter NanoLuc (Nluc) was obtained from Promega. TheSYP-Nluc reporter iPSC line was established by inserting SYP-Nluc fusionreporter in the AAVS1 site by the TALEN technology (Qian et. al, 2014).Astrocytes were differentiated from the isogenic iPSC lines D90D andD90A for 6 months by the protocol established in our lab (Krencik et.al, 2011). The astrocytes were plated at 1×10⁴ cells/well in white96-well plates (Greiner Bio-one) and cultured in the astrocyte medium(DMEM, 10% FBS) for 7 days. The D90A MNX1+MNs derived from SYP-Nlucreporter iPSC line were then plated at 1×10⁴ cells/well on astrocytesand cultured in a nutrition deficient medium (DMEM/F12, Neurobasalmedium at 1:1, 1×N2, 0.5 μM RA, 0.1 μM Purmorphamine and 0.1 μM CompoundE). For testing the compounds, Riluzole (50 μM, Torcris), Kenpaullone (5μM, Tocris) and EphA inhibitor (50 μM, Calbiochem) were added in themedium. After coculturing for 10 days, the Nluc activity was detected byNano-Glo® Luciferase Assay (Promega) according to the manufacturer'sinstructions.

Statistical Analyses

For quantifications, experiments were performed at least in triplicates.Statistical significance was assessed using one-way ANOVA followed byTukey's test. Data were presented as mean±SEM.

TABLE 1 Human pluripotent stem cell lines used in this study Cell lineDiagnosis Gender Age ESC/iPSC H9 normal female blastocyst ESC IMR90normal female fetal iPSC D90A ALS (SOD1 mutation) female 50 iPSC A4V ALS(SOD1 mutation) female 65 iPSC SMA13 SMA male 3 iPSC SMA232 SMA male 0.6iPSC

TABLE 2 Primers used in qPCR analysis Primer name sequenceSMN full length forward CACCACCTCCCATATGTCCAGATT (SEQ ID NO: 1)SMN full length reverse GAATGTGAGCACCTTCCTTCTTT (SEQ ID NO: 2)SMN total forward ATGAGCTGTGAGAAGGGTGTTG (SEQ ID NO: 3)SMN total reverse TTGCCACATACGCCTCACATAC (SEQ ID NO: 4) NEFL forwardTTTCACTCTTTGTGGTCCTCA (SEQ ID NO: 5) NEFL reverse AGACCCTGGAAATCGAAGC(SEQ ID NO: 6) GAPDH forward CTCTCTGCTCCTCCTGTTCGAC (SEQ ID NO: 7)GAPDH reverse TGAGCGATGTGGCTCGGCT (SEQ ID NO: 8)

We claim:
 1. A method for expanding and maintaining a population ofNKX2.2⁺ hindbrain serotonergic neural progenitors, the method comprisingculturing NKX2.2⁺ hindbrain serotonergic neural progenitors in a culturemedium comprising a Wnt signaling pathway agonist, an inhibitor of thebone morphogenetic protein (BMP) signaling pathway, an inhibitor of thetransforming growth factor beta (TGFβ) signaling pathway, a Notchsignaling pathway agonist and a SHE signaling pathway agonist, wherein apopulation of NKX2.2⁺ hindbrain serotonergic neural progenitors ismaintained for at least 5 passages.
 2. The method of claim 1, whereinthe method further comprises detecting the expression of at least one ofSOX1, SOX2, NESTIN, N-Cadherin, and Ki67 in the NKX2.2⁺ hindbrainserotonergic neural progenitors.
 3. The method of claim 2, wherein themethod further comprises detecting expression of at least one of GBX2,KROX20, HOXA1-4, and HOXB1-4, and substantially no expression offorebrain, spinal cord, or midbrain markers in the NKX2.2⁺ hindbrainserotonergic neural progenitors.
 4. The method of claim 1, wherein theWnt signaling pathway agonist is a GSK3 inhibitor selected from thegroup consisting of CHIR99021 and 6-bromo-iridium-3′-oxime.
 5. Themethod of claim 1, wherein the BMP signaling pathway inhibitor isselected from the group consisting of DMH-1, Dorsomorphin, andLDN-193189.
 6. The method of claim 1, wherein the Notch signalingpathway agonist is a histone deacetylase (HDAC) inhibitor selected fromthe group consisting of valproic acid (VPA), suberoyl bis-hydroxamicacid (SAHA), and sodium butyrate.
 7. The method of claim 1, wherein theTGFβ signaling pathway inhibitor is selected from the group consistingof SB431542, SB505124, and A83-01.
 8. The method of claim 1, wherein theculture medium comprises CHIR99021, DMH-1, SB431542, VPA, andpurmorphamine.
 9. The method of claim 8, wherein the culture mediumcomprises about 1 μM to 3 μM CHIR99021; about 1 μM to 5 μM DMH-1; about1 μM to 5 μM SB431542; about 0.2 μM-2 μM VPA; and about 0.1 μM to 1 μMpurmorphamine.
 10. The method of claim 1, wherein the NKX2.2⁺ hindbrainserotonergic neural progenitors are obtained from a human embryo.